Eomes expression identifies the early bone marrow precursor to classical NK cells.

Natural killer (NK) cells traffic through the blood and mount cytolytic and interferon-γ (IFNγ)-focused responses to intracellular pathogens and tumors. Type 1 innate lymphoid cells (ILC1s) also produce type 1 cytokines but reside in tissues and are not cytotoxic. Whether these differences reflect discrete lineages or distinct states of a common cell type is not understood. Using single-cell RNA sequencing and flow cytometry, we focused on populations of TCF7+ cells that contained precursors for NK cells and ILC1s and identified a subset of bone marrow lineage-negative NK receptor-negative cells that expressed the transcription factor Eomes, termed EomeshiNKneg cells. Transfer of EomeshiNKneg cells into Rag2-/-Il2rg-/- recipients generated functional NK cells capable of preventing metastatic disease. By contrast, transfer of PLZF+ ILC precursors generated a mixture of ILC1s, ILC2s and ILC3s that lacked cytotoxic potential. These findings identified EomeshiNKneg cells as the bone marrow precursor to classical NK cells and demonstrated that the NK and ILC1 lineages diverged early during development.

Mesenchymal stem cell: keystone of the hematopoietic stem cell niche and a stepping-stone for regenerative medicine.

Mesenchymal stem cells (MSCs) are self-renewing precursor cells that can differentiate into bone, fat, cartilage, and stromal cells of the bone marrow. Recent studies suggest that MSCs themselves are critical for forming a niche that maintains hematopoietic stem cells (HSCs). The ease by which human MSC-like and stromal progenitor cells can be isolated from the bone marrow and other tissues has led to the rapid development of clinical investigations exploring their anti-inflammatory properties, tissue preservation capabilities, and regenerative potential. However, the identity of genuine MSCs and their specific contributions to these various beneficial effects have remained enigmatic. In this article, we examine the definition of MSCs and discuss the importance of rigorously characterizing their stem cell activity. We review their role and that of other putative niche constituents in the regulation of bone marrow HSCs. Additionally, how MSCs and their stromal progeny alter immune function is discussed, as well as potential therapeutic implications.

Decisions about dendritic cells: past, present, and future.

A properly functioning adaptive immune system signifies the best features of life. It is diverse beyond compare, tolerant without fail, and capable of behaving appropriately with a myriad of infections and other challenges. Dendritic cells are required to explain how this remarkable system is energized and directed. I frame this article in terms of the major decisions that my colleagues and I have made in dendritic cell science and some of the guiding themes at the time the decisions were made. As a result of progress worldwide, there is now evidence of a central role for dendritic cells in initiating antigen-specific immunity and tolerance. The in vivo distribution and development of a previously unrecognized white cell lineage is better understood, as is the importance of dendritic cell maturation to link innate and adaptive immunity in response to many stimuli. Our current focus is on antigen uptake receptors on dendritic cells. These receptors enable experiments involving selective targeting of antigens in situ and new approaches to vaccine design in preclinical and clinical systems.

Immune cell compartmentalization for brain surveillance and protection.

For decades, it was commonly accepted that the brain is secluded from peripheral immune activity and is self-sufficient for its maintenance and repair. This simplistic perception was based on the presence of resident immune cells, the microglia, and barrier systems within the brain, and the assumption that the central nervous system (CNS) lacks lymphatic drainage. This view was revised with the discoveries that higher functions of the CNS, homeostasis and repair are supported by peripheral innate and adaptive immune cells. The findings of bone marrow-derived immune cells in specialized niches, and the renewed observation that a lymphatic drainage system exists within the brain, further contributed to this revised model. In this Review, we describe the immune niches within the brain, the contribution of professional immune cells to brain functions, the bidirectional relationships between the CNS and the immune system and the relevance of immune components to brain aging and neurodegenerative diseases.

Single-cell proteo-genomic reference maps of the hematopoietic system enable the purification and massive profiling of precisely defined cell states.

Single-cell genomics technology has transformed our understanding of complex cellular systems. However, excessive cost and a lack of strategies for the purification of newly identified cell types impede their functional characterization and large-scale profiling. Here, we have generated high-content single-cell proteo-genomic reference maps of human blood and bone marrow that quantitatively link the expression of up to 197 surface markers to cellular identities and biological processes across all main hematopoietic cell types in healthy aging and leukemia. These reference maps enable the automatic design of cost-effective high-throughput cytometry schemes that outperform state-of-the-art approaches, accurately reflect complex topologies of cellular systems and permit the purification of precisely defined cell states. The systematic integration of cytometry and proteo-genomic data enables the functional capacities of precisely mapped cell states to be measured at the single-cell level. Our study serves as an accessible resource and paves the way for a data-driven era in cytometry.

Macrophages, rather than DCs, are responsible for inflammasome activity in the GM-CSF BMDC model.

Inflammasomes are one of the most important mechanisms for innate immune defense against microbial infection but are also known to drive various inflammatory disorders via processing and release of the cytokine IL-1ß. As research into the regulation and effects of inflammasomes in disease has rapidly expanded, a variety of cell types, including dendritic cells (DCs), have been suggested to be inflammasome competent. Here we describe a major fault in the widely used DC-inflammasome model of bone marrow-derived dendritic cells (BMDCs) generated with the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). We found that among GM-CSF bone marrow-derived cell populations, monocyte-derived macrophages, rather than BMDCs, were responsible for inflammasome activation and IL-1ß secretion. Therefore, GM-CSF bone marrow-derived cells should not be used to draw conclusions about DC-dependent inflammasome biology, although they remain a useful tool for analysis of inflammasome responses in monocytes-macrophages.

A Genome-wide CRISPR Screen in Primary Immune Cells to Dissect Regulatory Networks.

Finding the components of cellular circuits and determining their functions systematically remains a major challenge in mammalian cells. Here, we introduced genome-wide pooled CRISPR-Cas9 libraries into dendritic cells (DCs) to identify genes that control the induction of tumor necrosis factor (Tnf) by bacterial lipopolysaccharide (LPS), a key process in the host response to pathogens, mediated by the Tlr4 pathway. We found many of the known regulators of Tlr4 signaling, as well as dozens of previously unknown candidates that we validated. By measuring protein markers and mRNA profiles in DCs that are deficient in known or candidate genes, we classified the genes into three functional modules with distinct effects on the canonical responses to LPS and highlighted functions for the PAF complex and oligosaccharyltransferase (OST) complex. Our findings uncover new facets of innate immune circuits in primary cells and provide a genetic approach for dissection of mammalian cell circuits.

Caveolin-1-dependent nanoscale organization of the BCR regulates B cell tolerance.

Caveolin-1 (Cav1) regulates the nanoscale organization and compartmentalization of the plasma membrane. Here we found that Cav1 controlled the distribution of nanoclusters of isotype-specific B cell antigen receptors (BCRs) on the surface of B cells. In mature B cells stimulated with antigen, the immunoglobulin M BCR (IgM-BCR) gained access to lipid domains enriched for GM1 glycolipids, by a process that was dependent on the phosphorylation of Cav1 by the Src family of kinases. Antigen-induced reorganization of nanoclusters of IgM-BCRs and IgD-BCRs regulated BCR signaling in vivo. In immature Cav1-deficient B cells, altered nanoscale organization of IgM-BCRs resulted in a failure of receptor editing and a skewed repertoire of B cells expressing immunoglobulin-µ heavy chains with hallmarks of poly- and auto-reactivity, which ultimately led to autoimmunity in mice. Thus, Cav1 emerges as a cell-intrinsic regulator that prevents B cell-induced autoimmunity by means of its role in plasma-membrane organization.

Differentiation of type 1 ILCs from a common progenitor to all helper-like innate lymphoid cell lineages.

Innate lymphoid cells (ILCs) are a recently recognized group of lymphocytes that have important functions in protecting epithelial barriers against infections and in maintaining organ homeostasis. ILCs have been categorized into three distinct groups, transcriptional circuitry and effector functions of which strikingly resemble the various T helper cell subsets. Here, we identify a common, Id2-expressing progenitor to all interleukin 7 receptor-expressing, "helper-like" ILC lineages, the CHILP. Interestingly, the CHILP differentiated into ILC2 and ILC3 lineages, but not into conventional natural killer (cNK) cells that have been considered an ILC1 subset. Instead, the CHILP gave rise to a peculiar NKp46(+) IL-7Rα(+) ILC lineage that required T-bet for specification and was distinct of cNK cells or other ILC lineages. Such ILC1s coproduced high levels of IFN-γ and TNF and protected against infections with the intracellular parasite Toxoplasma gondii. Our data significantly advance our understanding of ILC differentiation and presents evidence for a new ILC lineage that protects barrier surfaces against intracellular infections.

S6K-STING interaction regulates cytosolic DNA-mediated activation of the transcription factor IRF3.

Cytosolic DNA-mediated activation of the transcription factor IRF3 is a key event in host antiviral responses. Here we found that infection with DNA viruses induced interaction of the metabolic checkpoint kinase mTOR downstream effector and kinase S6K1 and the signaling adaptor STING in a manner dependent on the DNA sensor cGAS. We further demonstrated that the kinase domain, but not the kinase function, of S6K1 was required for the S6K1-STING interaction and that the TBK1 critically promoted this process. The formation of a tripartite S6K1-STING-TBK1 complex was necessary for the activation of IRF3, and disruption of this signaling axis impaired the early-phase expression of IRF3 target genes and the induction of T cell responses and mucosal antiviral immunity. Thus, our results have uncovered a fundamental regulatory mechanism for the activation of IRF3 in the cytosolic DNA pathway.

Brain motor and fear circuits regulate leukocytes during acute stress.

The nervous and immune systems are intricately linked1. Although psychological stress is known to modulate immune function, mechanistic pathways linking stress networks in the brain to peripheral leukocytes remain poorly understood2. Here we show that distinct brain regions shape leukocyte distribution and function throughout the body during acute stress in mice. Using optogenetics and chemogenetics, we demonstrate that motor circuits induce rapid neutrophil mobilization from the bone marrow to peripheral tissues through skeletal-muscle-derived neutrophil-attracting chemokines. Conversely, the paraventricular hypothalamus controls monocyte and lymphocyte egress from secondary lymphoid organs and blood to the bone marrow through direct, cell-intrinsic glucocorticoid signalling. These stress-induced, counter-directional, population-wide leukocyte shifts are associated with altered disease susceptibility. On the one hand, acute stress changes innate immunity by reprogramming neutrophils and directing their recruitment to sites of injury. On the other hand, corticotropin-releasing hormone neuron-mediated leukocyte shifts protect against the acquisition of autoimmunity, but impair immunity to SARS-CoV-2 and influenza infection. Collectively, these data show that distinct brain regions differentially and rapidly tailor the leukocyte landscape during psychological stress, therefore calibrating the ability of the immune system to respond to physical threats.

TCF-1 upregulation identifies early innate lymphoid progenitors in the bone marrow.

The cellular and molecular events that drive the early development of innate lymphoid cells (ILCs) remain poorly understood. We show that the transcription factor TCF-1 is required for the efficient generation of all known adult ILC subsets and their precursors. Using novel reporter mice, we identified a new subset of early ILC progenitors (EILPs) expressing high amounts of TCF-1. EILPs lacked efficient T and B lymphocyte potential but efficiently gave rise to NK cells and all known adult helper ILC lineages, indicating that they are the earliest ILC-committed progenitors identified so far. Our results suggest that upregulation of TCF-1 expression denotes the earliest stage of ILC fate specification. The discovery of EILPs provides a basis for deciphering additional signals that specify ILC fate.

Batf3 maintains autoactivation of Irf8 for commitment of a CD8α(+) conventional DC clonogenic progenitor.

The transcription factors Batf3 and IRF8 are required for the development of CD8α(+) conventional dendritic cells (cDCs), but the basis for their actions has remained unclear. Here we identified two progenitor cells positive for the transcription factor Zbtb46 that separately generated CD8α(+) cDCs and CD4(+) cDCs and arose directly from the common DC progenitor (CDP). Irf8 expression in CDPs required prior autoactivation of Irf8 that was dependent on the transcription factor PU.1. Specification of the clonogenic progenitor of CD8α(+) cDCs (the pre-CD8 DC) required IRF8 but not Batf3. However, after specification of pre-CD8 DCs, autoactivation of Irf8 became Batf3 dependent at a CD8α(+) cDC-specific enhancer with multiple transcription factor AP1-IRF composite elements (AICEs) within the Irf8 superenhancer. CDPs from Batf3(-/-) mice that were specified toward development into pre-CD8 DCs failed to complete their development into CD8α(+) cDCs due to decay of Irf8 autoactivation and diverted to the CD4(+) cDC lineage.

Identification of cDC1- and cDC2-committed DC progenitors reveals early lineage priming at the common DC progenitor stage in the bone marrow.

Mouse conventional dendritic cells (cDCs) can be classified into two functionally distinct lineages: the CD8α(+) (CD103(+)) cDC1 lineage, and the CD11b(+) cDC2 lineage. cDCs arise from a cascade of bone marrow (BM) DC-committed progenitor cells that include the common DC progenitors (CDPs) and pre-DCs, which exit the BM and seed peripheral tissues before differentiating locally into mature cDCs. Where and when commitment to the cDC1 or cDC2 lineage occurs remains poorly understood. Here we found that transcriptional signatures of the cDC1 and cDC2 lineages became evident at the single-cell level from the CDP stage. We also identified Siglec-H and Ly6C as lineage markers that distinguished pre-DC subpopulations committed to the cDC1 lineage (Siglec-H(-)Ly6C(-) pre-DCs) or cDC2 lineage (Siglec-H(-)Ly6C(+) pre-DCs). Our results indicate that commitment to the cDC1 or cDC2 lineage occurs in the BM and not in the periphery.

Developmental Analysis of Bone Marrow Neutrophils Reveals Populations Specialized in Expansion, Trafficking, and Effector Functions.

Neutrophils are specialized innate cells that require constant replenishment from proliferative bone marrow (BM) precursors as a result of their short half-life. Although it is established that neutrophils are derived from the granulocyte-macrophage progenitor (GMP), the differentiation pathways from GMP to functional mature neutrophils are poorly defined. Using mass cytometry (CyTOF) and cell-cycle-based analysis, we identified three neutrophil subsets within the BM: a committed proliferative neutrophil precursor (preNeu) which differentiates into non-proliferating immature neutrophils and mature neutrophils. Transcriptomic profiling and functional analysis revealed that preNeu require the C/EBPε transcription factor for their generation from the GMP, and their proliferative program is substituted by a gain of migratory and effector function as they mature. preNeus expand under microbial and tumoral stress, and immature neutrophils are recruited to the periphery of tumor-bearing mice. In summary, our study identifies specialized BM granulocytic populations that ensure supply under homeostasis and stress responses.

The Majority of CD45- Ter119- CD31- Bone Marrow Cell Fraction Is of Hematopoietic Origin and Contains Erythroid and Lymphoid Progenitors.

The non-hematopoietic cell fraction of the bone marrow (BM) is classically identified as CD45- Ter119- CD31- (herein referred to as triple-negative cells or TNCs). Although TNCs are believed to contain heterogeneous stromal cell populations, they remain poorly defined. Here we showed that the vast majority of TNCs (∼85%) have a hematopoietic rather than mesenchymal origin. Single cell RNA-sequencing revealed erythroid and lymphoid progenitor signatures among CD51- TNCs. Ly6D+ CD44+ CD51- TNCs phenotypically and functionally resembled CD45+ pro-B lymphoid cells, whereas Ly6D- CD44+ CD51- TNCs were enriched in previously unappreciated stromal-dependent erythroid progenitors hierarchically situated between preCFU-E and proerythroblasts. Upon adoptive transfer, CD44+ CD51- TNCs contributed to repopulate the B-lymphoid and erythroid compartments. CD44+ CD51- TNCs also expanded during phenylhydrazine-induced acute hemolysis or in a model of sickle cell anemia. These findings thus uncover physiologically relevant new classes of stromal-associated functional CD45- hematopoietic progenitors.

SARS-CoV-2 infection induces long-lived bone marrow plasma cells in humans.

Long-lived bone marrow plasma cells (BMPCs) are a persistent and essential source of protective antibodies1-7. Individuals who have recovered from COVID-19 have a substantially lower risk of reinfection with SARS-CoV-28-10. Nonetheless, it has been reported that levels of anti-SARS-CoV-2 serum antibodies decrease rapidly in the first few months after infection, raising concerns that long-lived BMPCs may not be generated and humoral immunity against SARS-CoV-2 may be short-lived11-13. Here we show that in convalescent individuals who had experienced mild SARS-CoV-2 infections (n = 77), levels of serum anti-SARS-CoV-2 spike protein (S) antibodies declined rapidly in the first 4 months after infection and then more gradually over the following 7 months, remaining detectable at least 11 months after infection. Anti-S antibody titres correlated with the frequency of S-specific plasma cells in bone marrow aspirates from 18 individuals who had recovered from COVID-19 at 7 to 8 months after infection. S-specific BMPCs were not detected in aspirates from 11 healthy individuals with no history of SARS-CoV-2 infection. We show that S-binding BMPCs are quiescent, which suggests that they are part of a stable compartment. Consistently, circulating resting memory B cells directed against SARS-CoV-2 S were detected in the convalescent individuals. Overall, our results indicate that mild infection with SARS-CoV-2 induces robust antigen-specific, long-lived humoral immune memory in humans.

Activation of neutrophils by autocrine IL-17A-IL-17RC interactions during fungal infection is regulated by IL-6, IL-23, RORγt and dectin-2.

Here we identified a population of bone marrow neutrophils that constitutively expressed the transcription factor RORγt and produced and responded to interleukin 17A (IL-17A (IL-17)). IL-6, IL-23 and RORγt, but not T cells or natural killer (NK) cells, were required for IL-17 production in neutrophils. IL-6 and IL-23 induced expression of the receptors IL-17RC and dectin-2 on neutrophils, and IL-17RC expression was augmented by activation of dectin-2. Autocrine activity of IL-17A and its receptor induced the production of reactive oxygen species (ROS), and increased fungal killing in vitro and in a model of Aspergillus-induced keratitis. Human neutrophils also expressed RORγt and induced the expression of IL-17A, IL-17RC and dectin-2 following stimulation with IL-6 and IL-23. Our findings identify a population of human and mouse neutrophils with autocrine IL-17 activity that probably contribute to the etiology of microbial and inflammatory diseases.

Diversification of TAM receptor tyrosine kinase function.

The clearance of apoptotic cells is critical for both tissue homeostasis and the resolution of inflammation. We found that the TAM receptor tyrosine kinases Axl and Mer had distinct roles as phagocytic receptors in these two settings, in which they exhibited divergent expression, regulation and activity. Mer acted as a tolerogenic receptor in resting macrophages and during immunosuppression. In contrast, Axl was an inflammatory response receptor whose expression was induced by proinflammatory stimuli. Axl and Mer differed in their ligand specificities, ligand-receptor complex formation in tissues, and receptor shedding upon activation. These differences notwithstanding, phagocytosis by either protein was strictly dependent on receptor activation triggered by bridging of TAM receptor-ligand complexes to the 'eat-me' signal phosphatidylserine on the surface of apoptotic cells.

Plasma cells require autophagy for sustainable immunoglobulin production.

The role of autophagy in plasma cells is unknown. Here we found notable autophagic activity in both differentiating and long-lived plasma cells and investigated its function through the use of mice with conditional deficiency in the essential autophagic molecule Atg5 in B cells. Atg5(-/-) differentiating plasma cells had a larger endoplasmic reticulum (ER) and more ER stress signaling than did their wild-type counterparts, which led to higher expression of the transcriptional repressor Blimp-1 and immunoglobulins and more antibody secretion. The enhanced immunoglobulin synthesis was associated with less intracellular ATP and more death of mutant plasma cells, which identified an unsuspected autophagy-dependent cytoprotective trade-off between immunoglobulin synthesis and viability. In vivo, mice with conditional deficiency in Atg5 in B cells had defective antibody responses, complete selection in the bone marrow for plasma cells that escaped Atg5 deletion and fewer antigen-specific long-lived bone marrow plasma cells than did wild-type mice, despite having normal germinal center responses. Thus, autophagy is specifically required for plasma cell homeostasis and long-lived humoral immunity.